Human cellular model systems of β-thalassemia enable in-depth analysis of disease phenotype

β-thalassemia is a prevalent genetic disorder causing severe anemia due to defective erythropoiesis, with few treatment options. Studying the underlying molecular defects is impeded by paucity of suitable patient material. In this study we create human disease cellular model systems for β-thalassemia by gene editing the erythroid line BEL-A, which accurately recapitulate the phenotype of patient erythroid cells. We also develop a high throughput compatible fluorometric-based assay for evaluating severity of disease phenotype and utilize the assay to demonstrate that the lines respond appropriately to verified reagents. We next use the lines to perform extensive analysis of the altered molecular mechanisms in β-thalassemia erythroid cells, revealing upregulation of a wide range of biological pathways and processes along with potential novel targets for therapeutic investigation. Overall, the lines provide a sustainable supply of disease cells as research tools for identifying therapeutic targets and as screening platforms for new drugs and reagents.


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The proteomic data generated in this study have been deposited in the PRIDE database under accession code PXD044730, and the processed data uploaded as a Supplementary Data File. Data underlying the graphs presented in each figure are provided in the Source Data file.
Fully anonymised samples of peripheral blood mononuclear cells were obtained as a waste product from donor blood of three independent donors, and from blood samples of two beta thalassemia patients. As the ethics stipulates use of anonymised samples, it was not possible to report on sex and gender in this study.
Please specify the socially constructed or socially relevant categorization variable(s) used in your manuscript and explain why they were used. Please note that such variables should not be used as proxies for other socially constructed/relevant variables (for example, race or ethnicity should not be used as a proxy for socioeconomic status). Provide clear definitions of the relevant terms used, how they were provided (by the participants/respondents, the researchers, or third parties), and the method(s) used to classify people into the different categories (e.g. self-report, census or administrative data, social media data, etc.) Please provide details about how you controlled for confounding variables in your analyses.
Blood donors can donate once they have reached their 17th birthday. 1st time donors can donate up to their sixty-sixth birthday. Regular donors (i.e. those who give at least one donation in a two year period) can continue to donate beyond 70 years, provided they remain otherwise fit and well. The overall donor base is also more balanced towards women. At the end of November 2019, NHSBT had 818,831 active donors, of whom 354,345 (43%) were male and 464,486 (57%) female. In UK largest proportion of donors are Caucasian The beta thalassmeia patients were both adults but further information is not available due to the samples being full anonymised. Beta thalassemia is not a sex linked disorder. Beta thalassemia most commonly affects people who are of Mediterranean (Greek, Italian and Middle Eastern) or Asian descent. It is also relatively common in people of African descent.
Recruitment for blood donors is performed by NHSBT. The beta thalassemia patients were recruited by their treating clinician based purely on availability of patients attending for blood transfusion and patient consent.
Adult CD34+ cells were isolated from LRS cones, with informed consent from all donor, and used in accordance with the Declaration of Helsinki and approved by the National Health Service Ethics Committee (reference number 08/H0102/06) and the Bristol Research Ethics Committee (reference 12/SW/0199). Whole blood from beta thalassemia patients was obtained under ethics board North of Scotland REC (18/NS/0005) with informed consent.
No sample size calculation was performed. Sample sizes were based on previous studies and included at least three replicates.
There have been no data samples excluded.
At least three replicates were analysed per group. All attempts at replication were successful.
There was no randomization since replicate groups were chosen according to their phenotype.

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We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. The investigators of this study were not blinded for analysis of flow cytometry data as the same gating was used for all groups. For microscopy, slides were counted blindly. Validations for the antibodies used for flow cytometry: Mouse anti-CD36-Vioblue conjugated antibody (Clone AC106,Miltenyi Biotec) is extensively validated by the vendor (Specificity and sensitivity assay tests) (https:// www.miltenyibiotec.com/GB-en/products/cd36-antibody-anti-human-ac106.html#conjugate=viogreen:size=100-tests-in-1-ml). Annexin V-FITC conjugated antibody Miltenyi Biotec) was validated by the vendor where Jurkat cells, cultured with staurosporine (50 nM) for 15 hours, were stained with Annexin V conjugates followed by staining with DAPI and analyzed by flow cytometry (https://www.miltenyibiotec.com/GB-en/products/annexin-v-conjugates.html#conjugate=fitc:size=100-tests-in-1-ml).